Three cDNA clones of approximately 300 nucleotides each and comprising the VP8 region of the VP4 protein of human rotavirus (HRV) strain KU, were constructed, inserted into the pGEMEX-1 plasmid and expressed in Escherichia coli. Immunoblot analysis of the corresponding peptides designated A (amino acid 1 to 105), B (amino acid 85 to 183), or C (amino acid 153 to 254), were performed using antisera to KU or Wa (VP4 serotype 1A), DS-1 (VP4 serotype 1B), or 1076 or M37 (VP4 serotype 2) rotavirus strains. These hyperimmune sera were prepared in guinea pigs inoculated with purified virus or expressed VP4 protein. The immunoblot analysis showed that epitopes located in fragment B were VP4 serotype and subtype specific and suggested that this fragment may be responsible for VP4 serotype and subtype specificity. In contrast, fragments A and C contained epitopes that were conserved among different human rotavirus VP4 serotypes. The high level of cross-reactivity observed in neutralization assay with antisera to the expressed VP8 protein may result from antibody to the conserved A and C fragments.